The present invention relates to the provision of the genetic sequence of the major grass pollen allergen Phl p 4. The invention also covers fragments, new combinations of partial sequences and point mutants having a hypoallergenic action. The recombinant DNA molecules and the derived polypeptides, fragments, new combinations of partial sequences and variants can be utilized for the therapy of pollen-allergic diseases. The proteins prepared by recombinant methods can be employed for the in vitro and in vivo diagnosis of pollen allergies.
Type 1 allergies are of importance worldwide. Up to 20% of the population in industrialized countries suffers from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are liberated from sources of various origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001).
The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensitized individuals. If two IgE molecules are cross linked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding clinical symptoms.
A distinction is made between major and minor allergens depending on the relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers.
In the case of timothy grass (Phleum pratense), Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesen and Lowenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al., 1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54). Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3), 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int. Arch. Allergy Immunol. 97: 287-294, Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198) and Phl p 13 (Suck et al., 2000, Clin. Exp. Allergy 30: 324-332; Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402) have hitherto been identified as major allergens.
Phl p 4 has been mentioned as a basic glycoprotein having a molecular weight of between 50 and 60 kDa (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268). The Phl p 4 molecule is trypsin-resistant (Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198), and 70-88% of grass pollen allergy sufferers have IgE antibodies against this molecule (Valenta et al., 1993, Int. Arch. Allergy Immunol. 97: 287-294; Rossi et al., 2001, Allergy 56:1180-1185; Mari, 2003, Clin. Exp. Allergy 33:43-51). Homologous molecules have been described from related grass species (Su et al., 1991, Clin. Exp. Allergy 21: 449-455; Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348; Jaggi et al., 1989, J. Allergy Clin. Immunol. 83: 845-852; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; 14-17). These homologous molecules of the Poaceae form allergen group 4, whose molecules have high immunological cross-reactivity with one another both with monoclonal mouse antibodies and with human IgE antibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Su et al., 1996, J. Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998, Clin. Exp. Allergy 28:799-807; Gavrovi-Jankulovi et al., 2000, Invest. Allergol. Clin. Immunol. 10 (6): 361-367; Stumvoll et al. 2002, Biol. Chem. 383: 1383-1396; Grote et al., 2002, Biol. Chem. 383: 1441-1445; Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130: 87-107; Mari, 2003, Clin. Exp. Allergy, 33 (1): 43-51).
In contrast to the above-mentioned major allergens of Phleum pratense (Phl p 1, Phl p 2/3, Phl 5a and 5b, Phl p 6 and Phl p 13), the primary structure of Phl p 4 has not yet been elucidated. Likewise, there is no complete sequence of molecules from group 4 from other grass species.
The determination of the N-terminal amino acid sequence was hitherto unsuccessful. However, the causes of this are not known. Fischer et al. (J. Allergy Clin. Immunol., 1996; 98: 189-198) assume N-terminal blocking, but were able to purify an internal peptide after degradation with lysyl endopeptidase and to determine its sequence: IVALPXGMLK (SEQ ID NO: 7).
This peptide has homologies to peptide sequences in the ragweed allergens Amb a1 and Amb a2 and similarities to sequences in proteins from maize (Zm58.2), tomato (lat 59, lat 56) and tobacco (G10) (Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198). For Lolium perenne, peptide fragments having the following sequence have been described for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQ ID NO: 8) and GLIEFPAGV (SEQ ID NO: 9) (Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348).
Peptides have likewise been obtained from the group 4 allergen from Dactylus glomerata by enzymatic degradation and sequenced: DIYNYMEPYVSK (P15, SEQ ID NO: 10), VDPTDYFGNEQ (P17, SEQ ID NO: 11), ARTAWVDSGAQLGELSY (P20, SEQ ID NO: 12) and GVLFNIQYVNYWFAP (P22, SEQ ID NO: 13) (Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072).
Peptides have also been obtained from the group 4 allergen of subtropical Bermuda grass (Cynodon dactylon) by proteolysis and sequenced: KTVKPLYHTP (S, SEQ ID NO: 14), KQVERDFLTSLTKDIPQLYLKS (V49L, SEQ ID NO: 15), TVKPLYIITPITAAMI (T33S, SEQ ID NO: 16), LRKYGTAADNVIDAKWDAQGRLL (T35L, SEQ ID NO: 17), KWQTVAPALPDPNM (P2, SEQ ID NO: 18), VTWIESVPYIPMGDK (V26L, SEQ ID NO: 19), GTVRDLLXRTSNIKAFGKY (L25L, SEQ ID NO: 20), TSNIKAFGKYKSDYVLEPIPKKS (T22L, SEQ ID NO: 21), YRDLDLGVNQWG (P3, SEQ ID NO: 22), SATPPTHRSGVLFNI (V20L, SEQ ID NO: 23), and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (V14L, SEQ ID NO: 24) (Liaw et al., 2001, Biochem. Biophys. Research Communication 280: 738-743).
However, these described peptide sequences for Phl p 4 and group 4 allergens have hitherto not resulted in the elucidation of the complete primary structure of group 4 allergens.
The object on which the present invention is based therefore comprised the provision of the complete DNA sequence of Phl p 4 and of a corresponding recombinant DNA on the basis of which the Phl p 4 allergen can be expressed as protein and made available for pharmacologically significant utilization as such or in modified form.
The numbers used above and below for nucleotide or amino acid sequences “SEQ ID NO” relate to the sequence protocol attached to the description.